灵芝漆酶在黑曲霉中的重组表达及发酵条件优化

The Recombinant Expression of Laccase from Ganoderma lucidum in Aspergillus niger and the Key Factors Affecting Enzyme Production by Fermentation

【目的】在黑曲霉(A.niger)中重组表达灵芝L菌漆酶基因,并对其发酵条件进行优化,旨在获得一株高产灵芝漆酶的黑曲霉基因工程菌。【方法】从灵芝L菌克隆漆酶基因Lac-L,并根据黑曲霉密码子偏好性进行优化。选用黑曲霉糖化酶启动子(PglaA)、信号肽及终止子为表达原件,以米曲霉pyrG基因为筛选标记,构建漆酶表达盒,采用聚乙二醇-CaCl法将其转化至黑曲霉X1(ΔpyrG)原生质体中。对转化子进行PCR验证及固态发酵酶活测定,筛选高产漆酶重组菌株,并对阳性转化子固态发酵培养基碳源、无机与有机氮源比例、Cu^(2+)浓度及发酵时间进行优化。【结果】经尿嘧啶缺陷及含2,2′-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt),ABTS)培养基筛选获得25株重组漆酶转化子,酶活复筛获得灵芝漆酶异源表达6号重组菌株,发酵72 h酶活可达3532.6 U/kg,宿主菌株的酶活仅为58.37 U/kg。重组6号菌优化后的最适固态发酵条件为:以麸皮为基础培养基,添加2%麦芽糖、2%(NH)SO、0.25 g/L Cu^(2+)SO,发酵60 h时重组菌株产漆酶活力最高,达到6255.47 U/kg。【结论】在黑曲霉中成功实现灵芝L菌漆酶基因Lac-L的异源表达。

【Objective】 The aim of this study was to recombine and express laccase gene of Ganoderma lucidum L strain in Aspergillus niger(A.niger),and optimize its fermentation conditions, so as to obtain a genetic engineering strain with high yield of G.lucidum laccase.【Method】 Lac-L was cloned from G.lucidum L and optimized according to codon preference of A.niger.The promoter, signal peptide and terminator of A.niger glucoamylase(glaA) were selected as the expression source, and the pyrG gene of Aspergillus oryzae was selected as the screening marker.The laccase expression cassette was constructed and transformed into A.niger X1(ΔpyrG) protoplast by polyethylene glycol-CaClmethod.PCR verification and enzyme activity determination of the transformants were performed to screen recombinant strains with high laccase production, and the carbon source, the ratio of inorganic and organic nitrogen sources, the concentration of Cu^(2+)and fermentation time of the solid-state fermentation medium for positive transformants were optimized.【Result】 Twenty-five recombinant laccase transformants were screened by uracil deficiency and 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt)(ABTS) medium, and the heterologous expression strain 6 of G.lucidum laccase was obtained by enzyme activity rescreening.The enzyme activity reached 3 532.6 U/kg after 72 h fermentation, while it was only 58.37 U/kg in the host strain.The optimal solid-state fermentation conditions for recombinant strain 6 were as follows: Wheat bran was used as the basic medium, adding 2% maltose, 2%(NH4)SOand 0.25 g/L Cu^(2+)SO,and the laccase activity of the recombinant strain reached 6 255.47 U/kg after 60 h of fermentation.【Conclusion】 The heterologous expression of laccase gene Lac-L from G.lucidum L strain was successfully realized in A.niger.